Hydrophobic amino acid residues are critical for the immunodominant epitope of the goodpasture autoantigen - A molecular basis for the cryptic natureof the epitope

Goodpasture (GP) autoimmune disease is caused by autoantibodies to type IV collagen that bind to the glomerular basement membrane, causing rapidly pro gressing glomerulonephritis. The immunodominant GP(A) autoepitope is encomp assed by residues 17-31 (the E-A region) within the noncollagenous (NC1) do main of the alpha3(IV) chain. The GP epitope is cryptic in the NC1 hexamer complex that occurs in the type IV collagen network found in tissues and in accessible to autoantibodies unless the hexamer dissociates. In contrast, t he epitope for the Mab3 monoclonal antibody is also located within the E-A region, but is fully accessible in the hexamer complex. In this study, the identity of residues that compose the GP(A) autoepitope was determined, and the molecular basis of its cryptic nature was explored. This was achieved using site-directed mutagenesis to exchange the alpha3(IV) residues in the E-A region with the corresponding residues of the homologous but non-immuno reactive alpha1(IV) NC1 domain and then comparing the reactivity of the mut ated chimeras with GP(A) and Mab3 antibodies. It was shown that three hydro phobic residues (Ala(18), Ile(19), and Val(27)) and Pro(28) are critical fo r the GP(A) autoepitope, whereas two hydrophilic residues (Ser(21) and Ser( 31)) along with pro(28) are critical for the Mab3 epitope. These results su ggest that the cryptic nature of the GP(A) autoepitope is the result of qua ternary interactions of the alpha3, alpha4, and alpha5 NC1 domains of the h examer complex that bury the one or more hydrophobic residues. These findin gs provide critical information for understanding the etiology and pathogen esis of the disease as well as for designing drugs that would mimic the epi tope and thus block the binding of GP autoantibodies to autoantigen.